It is known that several species and strains of the genus Amycolatopsis synthesize compounds which are produced on an industrial scale. They make a variety of extracellular enzymes such as amylases, cellulases and proteases as well as secondary metabolites such as antibiotics and other pharmacologically active molecules. As a result, there has been an increasing interest in developing gene cloning methods for these organisms.
A. mediterranei and A. orientalis are known to produce rifamycins (Margalith and Pagani Appl. Microbiol 9:325-334, 1961) and vancomycins (Barna and Williams, Annu. Rev. Microbiol. 38:339-357, 1984) respectively. A recently introduced glycopeptide antibiotic Balhimycin which exhibits antibacterial activity against methicillin resistant Staphylococcus aureus strains has been isolated from Amycolatopsis sp. Y-86 21022DSM 5908 (Nadkarni et al. Antibiotics 47:334-341 1994). In addition, species of Amycolatopsis alongwith other closely related genera of the order Actinomycetales form the third major group of bacteria in terms of scales of antibiotic production.
Methods of gene cloning have been developed for several species of Streptomyces (Khosla et al. Molecular Microbiol. 6:3237-3239, 1992; Lal et al. CRC Critical Rev. Microbiol. 22 220-1257, 1996; Hopwood et al. Laboratory Manual, John Innes Foundation Norwich, 1985). Although A. mediterranei belongs to the same order (Actinomycetales) to which Streptomyces belongs, recombinant DNA techniques were not available for Amycolatopsis until recently. This was mainly due to the lack of any plasmid, suitable for vector development in Amycolatopsis. Furthermore, standard transformation procedures as used in Streptomyces. spp. are not applicable to these organisms and conjugation was the only technique for the introduction of DNA into these organisms (Schupp et al. J. Bacteriol. 121:128-136, 1975).
Several scientific groups have attempted to develop suitable cloning vectors for these organisms (Madon et al. Mol. Genet. 209:257-264 1987; Moretti et al. Plasmid 1:126-133 1985; Schupp and Divers, FEMS Microbiol. Lett. 36:159-162, 1986; Lal et al. CRC Critical Rev. Microbiol. 21:19-30, 1995) but this did not meet with any success. For instance, an indigenous plasmid pMEA100 (27.7 kb) was isolated from A. mediterranei LBG A3136 which could not be developed into a suitable cloning vector as it integrates into the chromosome (Zhu et al. Plasmid, 24:132-142, 1990). Although conjugative plasmid pMEA300 was subsequently isolated from A. methanolica NCIB 11946 (Vrijbloed et al. J. Bacteriol. 176:7086-7090, 1994; Vrijbloed et al. J. Bacteriol. 177:6666-6669, 1995) this plasmid also could not be suitably modified into a versatile cloning vector as it occurred both in the free and integrated states. For these reasons studies on genetic manipulations of A. mediterranei and related species remained hampered for quite sometime. We succeeded in the development of a preliminary cloning vector pRL1 (10.4 kb) for A. mediterranei (Lal et al. Appl. Environ. Microbiol. 57:665-671, 1991). The preliminary cloning vector pRL1 was constructed by cloning a 5.1 kb fragment of pA387 (29.6 kb), an indigenous plasmid of A. orientalis DSM 43387 into E. coli vector pDM10. pRL1 could be transformed into A. mediterranei and A. orientalis only by electroporation.
A disadvantage, inspite of the preliminary success in developing cloning vector pRL1 was that this vector required further improvements in terms of reduction in its size (pRL1 has a size of 10.4 kb). A further disadvantage was that the vector needed the addition of suitable selectable marker genes (which could be suitably expressed in different species and strains of Amycolatopsis) to make it an ideal cloning vector. In fact this was a very serious bottleneck in development of cloning vector for Amycolatopsis as many selectable marker genes have not yet become available for this group of organisms. Commonly available antibiotic resistance markers such as thiostrepton, hyromycin, viomycin, gentamycin etc. are not expressed suitably in Amycolatopsis, thus making them unsuitable for use. Not only this, even the kanamycin or neomycin resistance gene (km/neo) of pRL1 which functions effectively in E. coli was found not to be very suitable for selection of transformants in Amycolatopsis since different species or strains of Amycolatopsis were intrinsically resistant to kanamycin/neomycin. When transformants were selected under neomycin pressure, several mutant colonies appeared among transformants.
Another disadvantage was that the method of transformation through electroporation which we had developed in 1991 (Lal et al. Appl. Environ. Microbiol. 57,665-671, 1991) did not give transformation efficiency as high as required for a vector to be used for gene cloning. However, Kumar et al. (1994) (Kumar et al. Appl. Environ. Microbiol 60:4086-4093, 1994) used pRL1 cloning vector developed by us (Lal et al. Appl. Environ. Microbiol. 57:665-671, 1991) or derivatives derived from pRL1 to transform Nocardia lactamdurans (cephamycin producer) and reported an efficient method of transformation (based on the treatment of filaments with MgCl.sub.2 and CsCl as developed by Madon and Hutter (1991) (Madon and Hutter, J. Bacteriol. 173:6325-6331, 1991). In this method they transformed N. lactamdurans by direct treatment of mycelia with polyethylene glycol 1000 and CsCl. However, this method also did not work when tried in several strains of A. mediterranei and A. orientalis. Moreover the above method as reported by Kumar et al. (1994) is very cumbersome and time consuming.
Although Kumar et al. 1994 (Appl. Environ. Microbiol. 60 4086-4093 1994) also succeeded in introducing two markers: .alpha.-amylase genes of Streptomyces griseus and apramycin resistance gene of Streptomyces griesofuscus in pRL1 derivatives, these vectors again could not be used in A. mediterranei and A. orientalis for cloning as they lacked several features generally found in an ideal cloning vector.